In an effort to define immunodominant epitopes and to develop monospecific skin test antigens, T cell epitopes from the homologous 19 kDa proteins from Mycobacterium intracellulare and M. tuberculosis were defined with overlapping synthetic peptides. These two antigens, exhibiting 78% sequence homology, present an excellent opportunity for defining species-specific epitopes. The T cell activity of these mycobacterial peptides were first tested in vitro by challenging anti-M. tuberculosis, anti-M. avium, and anti-M. kansasii T cell lines and assaying for T cell proliferation. Six peptides derived from the 19 kDa M. tuberculosis antigen stimulated T cells. Two of these peptides were specifically active against only the M. tuberculosis T cell line. Four the peptides derived from the M. intracellulare 19 kDa antigen induced T cell proliferation, and two of these peptides had specific reactivity. The peptides were also assayed for their capacity to elicit delayed-type hypersensitivity (DTH) reactions in guinea pigs. All six of the tuberculosis peptides that were active in skin test assays elicited non-specific responses. However, one of nine active M. intracellulare peptides did elicit a monospecific DTH response. These experiments suggest that specific skin test reagents may be developed from synthetic peptides.